Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression.

Cyclophosphaty -45mide (Cyc) chemotherapy in young female cancer patients is associated with an increased risk of premature ovarian insufficiency (POI). This study was designed to investigate the protective role of melatonin (Mel) as an adjuvant against Cyc-induced POI. Female mice received a single intraperitoneal (i.p.) dose of Cyc (75 mg/kg). Mel protection was achieved in mice after i.p. injection of melatonin (50 mg/kg) every 24 h for four consecutive days prior to chemotherapy initiation and for 14 additional days. Ovarian reserve testing, hormonal assays for follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone (AMH), assessment of the oxidative stress status, and measurement of the relative expression of genes in PTEN/AKT/FOXO3a and mitochondrial apoptosis pathways were performed. The results showed that treatment with 50 mg/kg Mel significantly prevented Cyc-induced over-activation of primordial follicles by maintaining the plasma level of AMH and subsequently preventing litter size reduction in mice treated with Cyc chemotherapy. Importantly, Mel treatment significantly prevented ovarian granulosa cell loss by inhibiting the mitochondrial apoptotic pathway. Identifying the protective actions of Mel against Cyc-induced primordial follicle loss has important implications for fertility maintenance in young cancer patients undergoing chemotherapy.

Flavobacterium taihuense sp. nov., a bacterium isolated from lake sediment.

A novel bacterium, designated NAS39T, was isolated from the interfacial sediment of Taihu Lake in PR China and its taxonomic position was investigated by using a polyphasic approach. Cells of the isolate were Gram-stain-negative, aerobic, non-motile, catalase-positive, yellow and rod-shaped. Phylogenetic analyses based on 16S rRNA gene sequences supported that strain NAS39T formed a cluster within the genus Flavobacterium, and was most closely related to Flavobacterium laiguense LB2P30T (98.4 %), followed by Flavobacterium tiangeerense 0563T (97.4 %). The average nucleotide identity values between strain NAS39T and F. laiguense LB2P30T and F. tiangeerense 0563T were 82.5 and 75.3 %, respectively. The digital DNA-DNA hybridization values between strain NAS39T and F. laiguense LB2P30T and F. tiangeerense 0563T were 40.9 and 18.6 %, respectively. The genomic DNA G+C content was 34.1 mol%. The major respiratory quinone was menaquinone-6. The dominant cellular fatty acids were iso-C15 : 0 and summed feature 3 comprising C16 : 1 ω7c/C16 : 1 ω6c. The polar lipids comprised phosphatidyl ethanolamine, two amino lipids, three amino phospholipids and two unidentified lipids. Based on the phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, strain NAS39T (=MCCC 1K06094T=KACC 22328T) represents a novel species of the genus Flavobacterium, for which the name Flavobacterium taihuense sp. nov. is proposed.

Rufibacter hautae sp. nov., a red-pigmented bacterium from freshwater lake sediment, and proposal of Rufibacter quisquiliarum as a latter heterotypic synonym of Rufibacter ruber.

A taxonomic identification using a polyphasic approach was performed on strain NBS58-1T, which was isolated from the interfacial sediment of Taihu Lake in China. Strain NBS58-1T was Gram-stain-negative, aerobic, non-spore-forming and catalase-positive. Phylogenetic analyses based on 16S rRNA gene and three housekeeping genes (rpoB, gyrB and dnaK) sequences supported the position that strain NBS58-1T should be classified within the genus Rufibacter. The 16S rRNA gene sequence of strain NBS58-1T possessed the highest similarity to Rufibacter sediminis H-1T (96.60 %), followed by Rufibacter glacialis MDT1-10-3T (96.17 %). And the ANI value between strain NBS58-1T and R. glacialis MDT1-10-3T was 79.3 %. The respiratory quinone was menaquinone 7 (MK-7). The major cellular fatty acids comprised iso-C15 : 0 and summed feature 3. Phosphatidylethanolamine, two unidentified phospholipids and four unidentified lipids were the main polar lipids. The genomic DNA G+C content was 51.3 mol%. Based on phenotypic features and phylogenetic position, a novel species with the name Rufibacter hautae sp. nov. is proposed. The type strain is NBS58-1T=(KACC 21309T=MCCC 1K04037T). We also proposed Rufibacter quisquiliarum as a latter heterotypic synonym of Rufibacter ruber.

Gemmobacter caeruleus sp. nov., a novel species originating from lake sediment.

An aerobic, Gram-stain-negative, non-spore-forming and rod-shaped bacterial strain, designated N8T, was isolated from the interfacial sediment of Taihu Lake in PR China. The strain formed white to blue colonies on R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain N8T represented a member of the genus Gemmobacter and was most closely related to Gemmobacter aquaticus A1-9T (97.97 %). The average nucleotide identity and digital DNA-DNAhybridization values between strain N8T and G. aquaticus A1-9T based on their whole genomes were 78.8 and 21.7 %, respectively. Q-10 was the main predominant ubiquinone. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C18 : 0 and C16 : 0. The G+C content of the genomic DNA was 66.1 mol%. The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one unidentified phospholipid, two unidentified glycolipids and two unidentified lipids. Based on its physiological, biochemical and chemotaxonomic characteristics, strain N8T represents a novel species of the genus Gemmobacter, for which the name Gemmobacter caeruleus sp. nov. is proposed. The type strain is N8T=(KACC 21307T=MCCC 1K04036T).

Altererythrobacter amylolyticus sp. nov., isolated from lake sediment.

An aerobic, motile, Gram-stain-negative bacterium, designated strain NS1T, was isolated from interfacial sediment from Taihu Lake, China. The strain formed yellow colonies on R2A medium. Cells were ovoid to rod-shaped and non-spore-forming. Growth occurred at 15-40 °C (optimum, 28 °C), at pH 5.0-10.5 (optimum, 6.5-7.5) and in the presence of 0-1 % (w/v) NaCl (optimum, 0 %). Phylogenetic trees based on 16S rRNA gene sequences showed that strain NS1T represented a member of the genus Altererythrobacter and had the highest sequence similarity to Altererythrobacter troitsensis CCTCC AB 2015180T (97.1 %). The average nucleotide identity value between strain NS1T and the closest related strain based on their genomes was 78.6 %. The predominant ubiquinone was Q-10. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified phospholipid, an unidentified glycolipid and six unidentified lipids. The genomic DNA G+C content was 66.6 mol%. On the basis of phenotypic and genotypic characteristics, strain NS1T represents a novel species of the genus Altererythrobacter, for which the name Altererythrobacter amylolyticus sp. nov. is proposed. The type strain is NS1T (=CGMCC 1.13679T=NBRC 113553T).

Effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation implantation dysfunction.

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied. The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG), followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later. Then the female mice were mated with male at a ratio of 2:1 in the same cage at 6:00 p.m. The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time. Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m. of the next day. Fifty successfully pregnant mice were equally randomly divided into 5 groups: normal control pregnant group (NC), COH implantation dysfunction model group (COH), low dosage of Bu-Shen-An-Tai recipe group (LOW), middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH). Then from day 1, the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m. for 5 consecutive days. The concentrations of 17ß-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA). The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM). The histopathological changes of ovarian tissues were observed by HE staining. The number of atretic follicles and pregnant corpus luteum were also recorded. TUNEL was applied to measure apoptotic cells of ovarian tissues. Western blotting was used to detect the protein expression of apoptosis- related factors like Bax, Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice. The results showed that ovarian weight, the concentrations of E2 and P4, the number of atretic follicles and pregnant corpus luteum, as well as the apoptosis of granulosa cells were significantly increased in the COH group. The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased, agglutinated, aggregated or crescent-shaped. The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis. After the treatment with different doses of Bu-Shen-An-Tai recipe, the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM. Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced. The lipid dropltes were shown in a circluar or oval shape. The protein expression levels of Bax and cleaved-caspase-3 were decreased, and the expression of Bcl-2 protein was increased after treatment. It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells, probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3, which contributes to the formation and maintenance of ovarian corpus luteum. It's helpful to promote the embryonic implantation, to reduce embryo loss and ultimately to improve the success rate of pregnancy.

Effect and Underlying Mechanism of Bu-Shen-An-Tai Recipe on Ovarian Apoptosis in Mice with Controlled Ovarian Hyperstimulation Implantation Dysfunction / 华中科技大学学报（医学）（英德文版）

The effect and underlying mechanism of Bu-Shen-An-Tai recipe on ovarian apoptosis in mice with controlled ovarian hyperstimulation (COH) implantation dysfunction were studied.The COH implantation dysfunction model in mice was established by intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotrophin (PMSG),followed by 7.5 IU human chorionic gonadotrophin (HCG) 48 h later.Then the female mice were mated with male at a ratio of 2:l in the same cage at 6:00 p.m.The female mice from normal group were injected intraperitoneally with normal saline and mated at the corresponding time.Day 1 of pregnancy was recorded by examining its vaginal smears at 8:00 a.m.of the next day.Fifty successfully pregnant mice were equally randomly divided into 5 groups:normal control pregnant group (NC),COH implantation dysfunction model group (COH),low dosage of Bu-Shen-An-Tai recipe group (LOW),middle dosage of Bu-Shen-An-Tai recipe group (MID) and high dosage of Bu-Shen-An-Tai recipe group (HIGH).Then from day 1,the mice in different groups were respectively intragastrically given corresponding treatments at 9:00 a.m.for 5 consecutive days.The concentrations of 17β-estradiol (E2) and progesterone (P4) were determined by radioimmunoassay (RIA).The ultrastructural changes of ovarian tissues were observed by transmission electron microscope (TEM).The histopathological changes of ovarian tissues were observed by HE staining.The number of atretic follicles and pregnant corpus luteum were also recorded.TUNEL was applied to measure apoptotic cells of ovarian tissues.Western blotting was used to detect the protein expression of apoptosis-related factors like Bax,Bcl-2 and cleaved-caspase-3 in ovarian tissue of mice.The results showed that ovarian weight,the concentrations of E2 and P4,the number of atretic follicles and pregnant corpus luteum,as well as the apoptosis of granulosa cells were significantly increased in the COH group.The ultrastructures of ovarian tissues in the COH group showed that chromatin in granulosa cells was increased,agglutinated,aggregated or crescent-shaped.The focal cavitation and the typical apoptotic bodies could be seen in granulosa cells in the late stage of apoptosis.After the treatment with different doses of Bu-Shen-An-Tai recipe,the ultrastructural changes of ovarian granulosa cells apoptosis were dramatically improved and even disappeared under TEM.Visible mitochondria and mitochondrial cristae were increased and vacuoles were significantly reduced.The lipid dropltes were shown in a circluar or oval shape.The protein expression levels of Bax and cleaved-caspase-3 were decreased,and the expression of Bcl-2 protein was increased after treatment.It was concluded that Bu-Shen-An-Tai recipe can inhibit the apoptosis of ovarian granulosa cells,probably by up-regulating the protein expression of Bcl-2 and down-regulating Bax and cleaved-caspase-3,which contributes to the formation and maintenance of ovarian corpus luteum.It's helpful to promote the embryonic implantation,to reduce embryo loss and ultimately to improve the success rate of pregnancy.

Effect and mechanism of Bushen Huoxue recipe on ovarian reserve in mice with premature ovarian failure.

The aim of the present study was to explore the effect and mechanism of Bushen Huoxue recipe (BHR) on ovarian reserve in mice with premature ovarian failure (POF). Mice were divided into 3 groups: normal group, model group and BHR group. Intraperitoneal injection of cyclophosphamide was performed to create the POF model. Primordial follicular (PDF) number, ovarian wet weight, ovarian index, and estrous cycle were analyzed to evaluate the effect of BHR on POF. Meanwhile, the mRNA and protein level of Mouse Vasa Homologue (MVH) in the bone marrow, peripheral blood and ovary were detected, to explore the underlying mechanism of the treatment efficacy of BHR on ovarian reserve. By the time of BHR treatment for 28 days, BHR increased the PDF number and shortened the estrous cycle of POF mice. BHR also decreased the mRNA level of MVH in the bone marrow, and increased mRNA and protein level of MVH in the ovary of POF mice. Our results demonstrated a treatment efficacy of BHR on POF mice, and revealed that BHR might repair the dysfunction of germline stem cells in the bone marrow, and thus to improve the ovarian reserve and enhance the ovarian function of POF mice through neo-oogenesis.

[Effect of Guishen Pill on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve].

OBJECTIVE: To study the effect of Guishen Pill (GSP) on expression levels of Oct-4, MVH, and Egr-1 in mice with diminished ovarian reserve (DOR). METHODS: Totally 40 female C57BL/6J mice were randomly divided into 4 groups, the normal control group, the model group, the GSP group, and the dehydroepiandrosterone (DHEA) group, 10 in each group. Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG), and prostaglandin F2α (PGF2α) were sequentially administrated to produce superovulation. The DOR model was established by exposing to ozone inhalation. Mice in the GSP group were intragastrically administered with GSP at 0.3 mL. Those in the DHEA group were intragastrically administered with DHEA at 0.3 mL. Equal volume of normal saline was intragastrically administered to mice in the normal control group and the model group. All mice wer treated for 21 days. Serum levels of estrogen (E2), progestogen (P), and anti-Müllerian hormone (AMH) were measured by ELISA. Changes of Oct-4, anti-AMH, and early growth response gene-1 (Egr-1) mRNA in ovaries were dtected by Real-time PCR. RESULTS: Compared with the model group, serum levels of E2, P, and AMH, as well as contents of estrogen receptor (ER), progestogen receptor (PR), MVH, and Oct-4 mRNA significantly increased in the GSP group and the DHEA group (P < 0.05). CONCLUSION: GSP could improve expression levels of Oct-4, MVH, and Egr-1 mRNA in DOR mice and their ovarian function.

Effects of Bu-Shen-An-Tai recipe and its two components on endometrial morphology during peri-implantation in superovulated mice.

The aim of this study was to investigate the effect of Bu-Shen-An-Tai recipe (BSATR) and its two components (Bushen recipe, and Huoxue recipe) on endometrial morphology during peri-implantation in superovulated mice. Mice were randomly divided into five groups, including the normal (N), model (M), Bushen (BS), Huoxue (HX) and Bu-Shen-An-Tai (BH) groups. The uteri were collected on day 4 of pregnancy, and the endometrium thickness, microvessel density (MVD) and number of pinopodes observed. Compared with the M group, the endometrial thickness in the BS, HX and BH groups was significantly increased and there was a significant difference in endometrial thickness between the BS and the BH groups. The mean MVD was significantly lower in the M group than in the N group, and there was a significant increase in MVD in the BS, HX and BH groups as compared with the M group. Compared with the M group, the pinopode scores in the endometrium were significantly increased in the HX and BH groups; and the BS group had significantly higher pinipode scores than the HX and BH groups. In conclusion, the results of the present study demonstrated that the recipes (Bushen, Huoxue and BSATR) could improve the endometrial environment by regulating the endometrial thickness, MVD and the number of pinopodes at the window of implantation. Moreover, the Huoxue recipe and the BSATR were more efficient than the Bushen recipe, with the BSATR tending to have the most beneficial effects.

[Measurement of night sky spectral radiation and analysis of spectral denoising].

In spectral radiation detection, spectral characteristics of the environment are completely different from standard light sources, so the output characteristics of optical imaging device is bound to bring greater error due to spectrum matching in imaging characteristic analysis. In the present paper, we measured moon-light and star-light spectral radiation using a portable CCD fiber spectrometer, and analyzed spectral characteristics, which provided a reference to correctly evaluate the imaging performance of optical imaging device.